Molecular biology of positive strand RNA viruses

Our current research apiary is destined to be built over early in 2018 with a new road development. We’ve therefore been planning and preparing a new site – increasing our capacity for both bees, storage and working area.

We are conducting a trial of coordinated Varroa treatment in conjunction with the beekeepers on the Isle of Arran. In late September DJE and Luke Woodford – the EastBIO-funded student leading the project – visited Arran to present an evening lecture on Varroa and DWV: Science and Practical Beekeeping.

Our studies exploit our understanding of changes in the virus population associated with Varroa transmission (rather than transmission from bee to bee during feeding) to monitor the ‘health’ of the colony.

The talk was followed by an extended question and answer session covering the project and more general aspects of beekeeping and Varroa control.

The Isle of Arran was looking fantastic as we crossed from Ardrossan on the CalMac ferry. Goatfell, the highest point on the Arran horseshoe ridge (see image above) is a great walk and thoroughly recommended.

Congratulations to Fadi Al Naji who recently completed the minor corrections to his PhD. thesis entitled “Isolation and analysis of recombinants from mixed virus infections of poliovirus using next generation sequencing (NGS) and bioinformatics“. Fadi was registered in the School of Life Sciences, University of Warwick and remained there to complete his studies after the rest of the Evanslab moved to St. Andrews. His external examiner was Professor Glyn Stanway, University of Essex (on the left below). Fadi is the good-looking one in the middle.

Just back from the excellent Europic 2016 meeting in Switzerland where Kirsten Bentley“knocked their socks off” with a talk on her recent analysis of the role of sequence identity and RNA structure in genetic recombination in enteroviruses.

Here’s what our friends in the Cameron and Vignuzzi labs tweeted …

Kirsten at Europic 2016

With thanks to Caroline, Urs and Laurent for running a great meeting …

The fidelity of the virus polymerase influences the rate of genetic recombination between viruses coinfecting the same cell. We used cell-based and new, biochemically-defined, assays to demonstrate that the viral polymerase is necessary and sufficient for the strand-transfer event of RNA virus recombination. Furthermore, the fidelity of the polymerase is critical in determining the efficiency with which recombination occurs; low fidelity polymerases exhibit high recombination rates, and vice versa.

Abstract

Genetic recombination in single-strand, positive-sense RNA viruses is a poorly understand mechanism responsible for generating extensive genetic change and novel phenotypes. By moving a critical cis-acting replication element (CRE) from the polyprotein coding region to the 3′ non-coding region we have further developed a cell-based assay (the 3′CRE-REP assay) to yield recombinants throughout the non-structural coding region of poliovirus from dually transfected cells. We have additionally developed a defined biochemical assay in which the only protein present is the poliovirus RNA dependent RNA polymerase (RdRp), which recapitulates the strand transfer events of the recombination process. We have used both assays to investigate the role of the polymerase fidelity and nucleotide turnover rates in recombination. Our results, of both poliovirus intertypic and intratypic recombination in the CRE-REP assay and using a range of polymerase variants in the biochemical assay, demonstrate that RdRp fidelity is a fundamental determinant of recombination frequency. High fidelity polymerases exhibit reduced recombination and low fidelity polymerases exhibit increased recombination in both assays. These studies provide the basis for the analysis of poliovirus recombination throughout the non-structural region of the virus genome and provide a defined biochemical assay to further dissect this important evolutionary process.

One of our colonies in the bee shed swarmed last week. The swarm ended up clustering around the entrance to the hive it had ‘escaped’ from. It was captured and rehoused successfully. The swarm in the picture is up to 5cm deep in places and probably contains 10-15,000 worker bees … and a single queen bee.

Swarmtastic!

Swarming is the natural way that a honey bee colony ‘reproduces’. The old queen and all of the older foragers leave the hive to establish a new colony. The remaining workers raise a new queen from an egg or young larva in the original hive, so generating two colonies from one. Swarming usually occurs in late Spring or early Summer.

Not as unconnected as you might think. The most numerous photosynthetic organisms on earth – the cyanobacteria – are infected by viruses (cyanhophages). Some of these cyanhophages carry components of the photosynthetic machinery and are thought to contribute to host cell photosynthesis. In a recent study on which we collaborated we show that virus-infected cyanobacteria are inhibited in their ability to fix CO2 (in contrast to uninfected cyanobacteria) whereas photosynthetic electron transport is unaltered. The cyanhophages therefore redirect photosynthesis to support phage development.

These results also have implications for our understanding of global warming. The reduction in CO2 fixation in the marine environment, as a consequence of these cyanophage infections, may be as much as 10%. The global warming calculations are based on assumptions of carbon fixation levels being directly linked to photosynthetic activity. We show that that this is incorrect and that CO2 fixation is likely overestimated in marine environments.

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus are the most numerous photosynthetic organisms on our planet. With a global population size of 3.6 × 1027, they are responsible for approximately 10% of global primary production. Viruses that infect Prochlorococcus and Synechococcus (cyanophages) can be readily isolated from ocean waters and frequently outnumber their cyanobacterial hosts. Ultimately, cyanophage-induced lysis of infected cells results in the release of fixed carbon into the dissolved organic matter pool. What is less well known is the functioning of photosynthesis during the relatively long latent periods of many cyanophages. Remarkably, the genomes of many cyanophage isolates contain genes involved in photosynthetic electron transport (PET) as well as central carbon metabolism, suggesting that cyanophages may play an active role in photosynthesis. However, cyanophage-encoded gene products are hypothesized to maintain or even supplement PET for energy generation while sacrificing wasteful CO2 fixation during infection. Yet this paradigm has not been rigorously tested. Here, we measured the ability of viral-infected Synechococcus cells to fix CO2 as well as maintain PET. We compared two cyanophage isolates that share different complements of PET and central carbon metabolism genes. We demonstrate cyanophage-dependent inhibition of CO2 fixation early in the infection cycle. In contrast, PET is maintained throughout infection. Our data suggest a generalized strategy among marine cyanophages to redirect photosynthesis to support phage development, which has important implications for estimates of global primary production.

Our snappily-titled manuscript “The Iflaviruses Sacbrood virus and Deformed wing virus evoke different transcriptional responses in the honeybee which may facilitate their horizontal or vertical transmission” has just been published in PeerJ. We analysed changes in the transcriptome following infection with deformed wing virus (DWV) and sacbrood virus, or DWV alone. We propose that the difference in expression we observed of the honeybee immune genes induced by SBV and DWV may be an evolutionary adaptation to the different predominant transmission routes used by these viruses .

On Thursday 14th January I’m talking at an evening meeting of Fife Beekeepers Association about the biology of Deformed Wing Virus and how our understanding of the virus should help devise more rational integrated pest management strategies. This is the first of several outreach events planned for 2016 in which our BBSRC-funded research on honeybee viruses will be discussed.

Not long now until the beekeeping season starts and we can get on with our planned field studies 🙂